Contents of Part 2- Immunocontraception 2.1 How does it work? 2.2 Has it been tried out? 2.3 Is it safe? 2.4 So should we try this vaccine out here?

2 PZP Immunocontraception ---------------------------------- Many people have sought to find a non-lethal alternative to culling deer. Such research as there has been has centred on using various forms of contraception to reduce the birth rate. While a fair amount has been published on this, it's difficult for a non-biochemist to trawl through the scientific literature to find out exactly what's what.

By far the most promising method for this is 'immunocontraception'. This means utilising the animals own immune system to prevent pregnancy. Of the various systems that have been devised, 'PZP' (porcine or pig zona pellucida) vaccination is the most widely advocated.

2.1 How does it work? ---------------------- Zona pellucida is a protein that surrounds mammalian egg cells. It forms a kind of jelly like layer around the egg cell. The principle of this vaccination is that by administering zona pellucida from pigs, the immune system will produce antibodies that bind to the egg and prevent the entry of sperm. The protein is very similar in different species of mammals, so in principle taking ZP from one species and using it in another can work. That said there is sufficient variation in this protein between species that we cannot assume a single vaccine will be applicable in any species until it has been tested.For example:

Differences between antigenic determinants of pig and cat zona pellucida proteins Jewgenow K, Rohleder M, Wegner I JOURNAL OF REPRODUCTION AND FERTILITY 119: (1) 15-23 MAY 2000 "Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization."

It is therefore premature to assume that the PZP vaccine as is will work on all species of deer.

Because the protein is so well conserved between species, the body isn't terribly good at recognising it as 'foreign'. Like in many other vaccinations, another compound must be added to give the immune system a helpful boost, to make it respond to the protein. This is called an adjuvant. Therefore, in any assessment of the safety and effectiveness of a vaccine, the complete mix that makes up the vaccine must be taken into account.

2.2 Has it been tried out? ------------------------- PZP has been piloted in a number of trials, on deer populations this has been done in the United States.

That PZP immunocontraception has worked well to reduce pregnancy rates in white-tailed deer. For example;

Effectiveness, reversibility, and serum antibody titers associated with immunocontraception in captive white-tailed deer Turner JW, Kirkpatrick JF, Liu IKM JOURNAL OF WILDLIFE MANAGEMENT 60: (1) 45-51 JAN 1996 "Our data demonstrate in white-tailed does that PZP vaccination: (1) can produce contraception for at least 1 breeding season, (2) yields greater efficacy with multiple injections than with 1 injection, (3) is reversible within 2 years in 75% of does, (4) is associated with elevated anti-PZP antibody titers, and (5) can produce a sustained antibody response through at least 1 breeding season. The titer data suggest that PZP vaccine in white-tailed deer behaves in a manner physiologically similar to PZP vaccine effects reported in several other species."

A number of trials are described in on-line sources, such as http://www.nist.gov/public_affairs/factsheet/deer.htm, and http://www.fairharbor.com/fhca_deer_immunocontr.htm.

2.3 Is it safe? ---------- When deciding whether to use such a vaccine, we must also take into account any potential side effects that it may have. This has also been assessed in a number of studies.

Immunocontraception of captive exotic species .2. Formosan sika deer (Cervus nippon taiouanus), axis deer (Cervus axis), Himalayan tahr (Hemitragus jemlahicus), Roosevelt elk (Cervus elaphus roosevelti), Reeves' muntjac (Muntiacus reevesi), and sambar deer (Cervus unicolor) Kirkpatrick JF, Calle PP, Kalk P, Liu IKM, Turner JW JOURNAL OF ZOO AND WILDLIFE MEDICINE 27: (4) 482-495 DEC 1996 "Two Formosan sika deer fawns, two axis deer fawns, one Roosevelt elk calf, and two sambar deer fawns whose mothers became pregnant after inoculation with PZP were born healthy. Animals pregnant when inoculations began included one Formosan sika, one axis deer, and three sambar deer. The sika deer delivered a healthy fawn, the axis deer delivered a stillborn fawn, one of the three sambar fawns was stillborn, and a second sambar fawn was abnormal. Six of 13 sambar deer fawns that were conceived after inoculation and were exposed to the PZP vaccine, adjuvants, and anti-PZP antibodies during gestation were born with a variety of weaknesses and health problems or were stillborn."

Potential consequences and problems with wildlife contraceptives Nettles VF REPRODUCTION FERTILITY AND DEVELOPMENT 9: (1) 137-143 1997 "Concern is expressed that the use of immunocontraception could create genetic changes in the target population that would influence disease resistance."

The concern discussed in this paper is complex. By administering a vaccine that prevents deer with stronger immune systems from breeding, it is feared that diseased, immunocompromised deer will breed at the expense of healthier deer, passing on greater susceptibility to disease to the next generation.

Behavioural changes, such as those highlighted in the study by McShea et al. above must also be taken into account. Another study looking at this was:

Behavioral response of free-ranging elk treated with an immunocontraceptive vaccine Heilmann TJ, Garrott RA, Cadwell LL, Tiller BL JOURNAL OF WILDLIFE MANAGEMENT 62: (1) 243-250 JAN 1998 "We tested the hypothesis that breeding behavior would be prolonged in PZP-treated cows due to repeated estrus cycling. Sexual interaction rates of treatment and control cows were similar during the normal breeding season (P = 0.96) but differed significantly during the postbreeding season (P < 0.001), when almost no sexual interactions were observed in control cows." "We recommend population-level experiments to further our understanding of the effects of contraceptive technologies on the behavior and dynamics of free-ranging ungulate herds."

It is clear from these studies that while this drug may be effective in limiting the number of births in a wild population, the side effects are as yet poorly understood, and before any population-wide application can be condoned, significant studies must be carried out with the specific deer species to ascertain the extent of any adverse effects.

The question of what scale of population of deer can be treated with PZP must also be addressed. Trials to date have concentrated on small scale, geographically isolated populations with no natural predators.

One of the better-known trials is detailed in this study.

Evaluating immunocontraception for managing suburban white-tailed deer in Irondequoit, New York Rudolph BA, Porter WF, Underwood HB JOURNAL OF WILDLIFE MANAGEMENT 64: (2) 463-473 APR 2000 "Our objectives were to estimate effort required to apply porcine zona pellucida (PZP) to individual deer and assess the utility of using immunocontraception to control growth of deer populations. The study was conducted in a 43-km(2) suburban community with about 400 deer. Effort per deer was measured as time required to capture and mark deer, and then to apply booster immunocontraceptive treatments by remote injection." "Immunocontraception has the best potential for holding suburban deer populations between 30 and 70% of ecological carrying capacity, but is likely to be useful only in localized populations when the number of females to be treated is small (e.g., less than or equal to 200 deer)."

A more general model is discussed in this study:

Effects of fertility control on populations of ungulates: General, stage-structured models Hobbs NT, Bowden DC, Baker DL JOURNAL OF WILDLIFE MANAGEMENT 64: (2) 473-491 APR 2000 "(1) More than 50% of fertile females will need to be maintained infertile to achieve meaningful reductions in ungulate numbers even when fertility rates are low (2) The relationship between the proportion of females maintained infertile and the steady state density is highly nonlinear. This means that small errors in estimating levels of infertility can lead to large errors in achieved density. It also means that managers should expect to see little change in steady-state density across a broad range of delivery rates. (3) The efficacy of fertility control as a management technique depends strongly on the persistence of the effect of the fertility control agent and the ability of managers to recognize previously treated animals. (4) Fertility control using long-lived agents can be more efficient than culling in regulating ungulate numbers. (5) Treating small populations with irreversible agents magnifies the likelihood of population extinction relative to treatment by culling. As with all techniques, managing population fertility must extend from a sound understanding of the influence of management actions on the state and dynamics of the population."

In plain English, this means that to effectively use this technology to maintain a stable, sutstainable population is fraught with difficulties. Very small errors in the rate of application of the vaccine could lead to large differences in population, and even with a very low fertility rate a large proportion of the females must be treated.

The published research to date does not advocate the widespread application of PZP, nor does it clearly demonstrate the effectiveness or safety of that vaccine in any of our native deer. Indeed, the recommendations from these studies are that this kind of treatment will only be useful for small, contained populations in which detailed population and physiological studies have been conducted. None of this applies to species of deer in the UK, where a lack of natural predation means that the overpopulation problem is more general, and appropriate physiological, efficacy and population studies have not been conducted.

2.4 So should we try this vaccine out here? ------------------------------------------------- As mentioned above, the decision as to whether to use a product like this must be based upon a sound understanding of the mode of action of the whole vaccine and the implication for conservation management.

When we look in greater depth into the makeup of the PZP vaccine, successful trials published to date have incorporated 'Freunds complete adjuvant' in their formulation. This is an oil-in water emulsion, containing in it dead cells of Mycobacterium tuberculosis. This is an intensely powerful immuno-stimulant, much used in research but not generally considered appropriate for use in final applications. For a good discussion of this, see;

Adjuvants And Their Modes Of Action Alexander J, Brewer JM LIVESTOCK PRODUCTION SCIENCE 42: (2-3) 153-162 Jun 1995 "Freund's Complete Adjuvant (FCA) has been used experimentally and does stimulate cellular immunity, but is unsuitable for human and veterinary use as it promotes, amongst other toxic side effects, local inflammation and granuloma formation at the site of injection."

The FDA has numerous documents governing the development of new vaccines. It is quite clear that the use of non-standard adjuvants is not encouraged (this taken from FDA Docket No. 97N-0029)

"Currently, the only adjuvants included in U.S. licensed vaccines are aluminum compounds. An adjuvant shall not be introduced into a product unless there is satisfactory evidence that it does not affect adversely the safety or potency of the product." "If a manufacturer contemplates incorporating an adjuvant other than aluminum compounds into combination vaccine formulations for preventable infectious diseases, discussions with CBER should be initiated early in product development regarding appropriate preclinical and clinical studies. Potential safety concerns for investigational adjuvants include injection site reactions (e.g., pain, induration, erythema, granuloma formation, sterile abscess formation), fever, other systemic adverse effects (e.g., nausea, malaise, headache), immune mediated events (e.g. anaphylaxis, uveitis, or arthritis), systemic chemical toxicity to tissues or organs, teratogenicity, and carcinogenicity."

The regulations governing this are more specifically laid out in CFR 610.15. "An adjuvant shall not be introduced into a product unless there is satisfactory evidence that it does not affect adversely the safety or potency of the product"

Considering the medical and veterinary implications of using FCA, the FDA would not approve the use of this drug formula.

Another significant factor in the development of a new vaccine is whether its use would have any wider implication for wildlife management or farming. Unfortunately, one of the effects of FCA is that as it contains dead Mycobacterium tuberculosis cells, it stimulates an immune response to them. This bacterium causes tuberculosis, meaning that treated animals will give a false-positive test for that condition (somewhat similar to the tuberculin test given prior to the BCG injection).

The importance of monitoring this condition is shown by this work:

The Epidemiology Of Mycobacterium-Bovis Infections In Animals And Man - A Review Oreilly LM, Daborn CJ TUBERCLE AND LUNG DISEASE 76: 1-46, Suppl. 1 AUG 1995 "Tuberculosis is primarily a respiratory disease and transmission of infection within and between species is mainly by the airborne route, Mycobacterium bovis, the cause of bovine-type tuberculosis, has an exceptionally wide host range, Susceptible species include cattle, humans, non-human primates, goats, cats, dogs, pigs, buffalo, badgers, possums, deer and bison, Many susceptible species, including man, are spillover hosts in which infection is not self-maintaining. In countries where there is transmission of infection from endemically infected wildlife populations to cattle or other farmed animals, eradication is not feasible and control measures must be applied indefinitely, Possible methods of limiting spread of infection from wildlife to cattle including the use of vaccines are outlined, The usefulness of DNA fingerprinting of M. bovis strains as an epidemiological tool and of BCG vaccination of humans and cattle as a control measure are reviewed."

Detection of tuberculosis in deer is already a difficult task;

Etiology, Pathogenesis And Diagnosis Of Mycobacterium-Bovis In Deer Griffin JFT, Buchan GS VETERINARY MICROBIOLOGY 40: (1-2) 193-205 May 1994 "The unique susceptibility of cervidae to mycobacteria in general has meant that diagnosis of tuberculosis in deer using conventional intradermal tuberculin tests may be unsatisfactory. Tuberculin testing in deer is more technically demanding than in cattle, with the cervical region being the most sensitive area. False positive skin reactions occur widely in non-diseased deer while seriously infected animals may be ''anergic'' and fail to react (false negative)."

We can say from these two studies that widespread false positive testing of deer for BTB in the UK would be catastrophic for the deer population. To remove our ability to detect this condition in a major wild host for the disease would mean culling on an unprecedented scale following the first outbreak of this disease, all wild deer testing positive would be culled. One only needs to think of the recent slaughter of badgers in the South West of England to see the severity of this issue.

From these papers showing the risk of false positive tuberculin tests, and from the information presented above on the side effects of Freunds complete adjuvant, it is hardly surprising that the Humane Society of the United States (HSUS), a strong advocator of this vaccine, came to this conclusion (http://www.hsus.org/whatnew/immuno071800.html)

".we must find a vaccine formulation that uses an adjuvant (an immune-system booster) that is acceptable to the U.S. Food and Drug Administration (FDA)-the current formulation, which incorporates Freund's Complete Adjuvant in the initial treatment, will not be approved by the FDA."